Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
  • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
  • C07K16/248—IL-6
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

• the present invention relates to a method for purifying anti-IL-6 receptor antibodies from a sample comprising said antibodies and impurities, through the use of a three-chromatographic columns procedure, including a chromatography on Fluorapatite-containing resin.
• the invention is also concerned with pharmaceutical compositions comprising the purified antibodies obtainable by the process of the invention.
• Process related impurities typically consist of HCPs (host cell proteins), DNA and rPA (residual protein A).
• HCPs are an important source of impurity and may represent a serious challenge due to their high complexity and heterogeneity in molecular mass, isoelectric point and structure. It is thus necessary to have therapeutic antibodies exhibiting very low levels of HCPs: a particular emphasis should be laid on the optimization of techniques to reduce HCPs during the downstream process (i.e. purification process). Furthermore the downstream process must be tailored in such a way as to comply with the quality produced by the corresponding upstream process. Product related impurities such as aggregates, antibody fragments must also be reduced to a minimal level for any kind of therapeutic antibodies.
• the present invention provides a method of purifying an anti-IL-6 receptor antibody from a sample containing the antibody to be purified and impurities, wherein the method comprises the following steps: (a) contacting the sample containing the anti-IL-6 receptor antibody and the impurities with a Protein A chromatography material under conditions such that the antibody binds to the resin and at least a portion of the impurities does not bind to the resin; (b) eluting the antibody from the Protein A chromatography material, in order to obtain an eluate; (c) loading the eluate of step (b) onto a first mixed mode chromatography material under conditions such that the antibody does not bind to the resin and at least a portion of the remaining impurities binds to the resin; (d) recovering the flowthrough containing the antibody under conditions such that said recovered flowthrough contains a lower level of impurities than the eluate of step (b), (e) loading the recovered flowthrough containing the antibody of step (d) onto a second mixed mode chromatography material
• the present invention also provides a method of obtaining an anti-IL-6 receptor antibody in a monomeric form, wherein the method comprises the following steps: (a) contacting the sample containing the antibody in monomeric form, aggregated form and/or fragmented form with a Protein A chromatography material under conditions such that the antibody binds to the resin and at least a portion of the aggregated form and fragmented form does not bind to the resin; (b) eluting the antibody in monomeric form from the Protein A chromatography material, in order to obtain an eluate; (c) loading the eluate of step (b) onto a first mixed mode chromatography material under conditions such that the antibody in monomeric form does not bind to the resin and at least a portion of the remaining aggregated form and fragmented form bind to the resin; (d) recovering the flowthrough containing the antibody in monomeric form under conditions such that said recovered flowthrough contains a lower level of aggregated form and fragmented form than the eluate of step (b), (e) loading the
• the anti-IL-6 receptor antibody to be purified according to the present invention is a monoclonal antibody, such as a monoclonal antibody selected from the group consisting of a chimeric antibody, a humanized antibody or a fully human antibody. It is preferably tocilizumab.
• the mixed mode supports of the present invention present a combination of two or more of the following functionalities such as cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.
• the mixed mode support for steps (c) is for instance selected from the group consisting of cation exchange and anion exchange, such as Capto-MMC and Capto-Adhere, and the mixed mode support of step (e) is selected from the group consisting of hydroxyfluorapatite or fluorapatite.
• methods of the invention advantageously keep product-related impurities (e.g. antibody aggregates and fragments), and process-related impurities including host cell proteins, DNA levels, and residual PA, within acceptable ranges.
• Methods of the invention also advantageously avoid substantial product loss and therefore allow purification of high quality antibodies at high yield.
• antibody and its plural form “antibodies”, includes, inter alia, polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2, Fab proteolytic fragments, and single chain variable region fragments (scFvs).
• antigen-binding fragments such as F(ab')2, Fab proteolytic fragments, and single chain variable region fragments (scFvs).
• scFvs single chain variable region fragments
• anti-IL-6 receptor antibody or "anti-IL-6R antibody” refers to an antibody, such as a monoclonal antibody, binding to the IL-6 receptor.
• the "anti-IL-6 receptor antibody” is preferably a humanized monoclonal antibody; preferably tocilizumab.
• the light chain of tocilizumab contains an amino acid sequence as defined in SEQ ID No.1 and the heavy chain of tocilizumab contains an amino acid sequence as defined in SEQ ID No.2.
• the term "monoclonal antibody” refers to an antibody that is a clone of a unique parent cell.
• humanized immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin.
• the non-human immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor” (humanization by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains onto human constant regions (chimerization).
• Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.
• a humanized immunoglobulin all parts of a humanized immunoglobulin, except possibly the CDRs and a few residues in the heavy chain constant region if modulation of the effector functions is needed, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
• biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
• fully human immunoglobulin refers to an immunoglobulin comprising both a human framework region and human CDRs. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a fully human immunoglobulin, except possibly few residues in the heavy chain constant region if modulation of the effector functions or pharmacokinetic properties are needed, are substantially identical to corresponding parts of natural human immunoglobulin sequences. In some instances, amino acid mutations may be introduced within the CDRs, the framework regions or the constant region, in order to improve the binding affinity and/or to reduce the immunogenicity and/or to improve the biochemical/biophysical properties of the antibody.
• recombinant antibody means antibody produced by recombinant techniques.
• Recombinant host cells for the production of antibodies include recombinant prokaryotic and eukarytic cells; preferably mammalian host cells, such as Chinese Hamster Ovary (CHO) cells (including CHO-S cells or CHO-k1 cells).
• CHO Chinese Hamster Ovary
• recombinant antibody therefore refers to an antibody produced in recombinant (e.g. mammalian) cells. Because of the relevance of recombinant DNA techniques in the generation of antibodies, one needs not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics.
• variable domain or constant region
• Changes in the constant region will, in general, be made in order to improve, reduce or alter characteristics, such as complement fixation (e.g. complement dependent cytotoxicity, CDC), interaction with Fc receptors, and other effector functions (e.g. antibody dependent cellular cytotoxicity, ADCC), pharmacokinetic properties (e.g. binding to the neonatal Fc receptor; FcRn).
• Changes in the variable domain will be made in order to improve the antigen binding characteristics.
• immunoglobulins may exist in a variety of other forms including, for example, single-chain or Fv, Fab, and (Fab')2 , as well as diabodies, linear antibodies, multivalent or multispecific hybrid antibodies.
• antibody portion refers to a fragment of an intact or a full-length chain or antibody, usually the binding or variable region. Said portions, or fragments, should maintain at least one activity of the intact chain / antibody, i.e. they are “functional portions” or “functional fragments”. Should they maintain at least one activity, they preferably maintain the target binding property.
• antibody portions include, but are not limited to, “single-chain Fv", “single- chain antibodies”, “Fv” or “scFv”. These terms refer to antibody fragments that comprise the variable domains from both the heavy and light chains, but lack the constant regions, all within a single polypeptide chain.
• a single-chain antibody further comprises a polypeptide linker between the VH and VL domains which enables it to form the desired structure that would allow for antigen binding.
• single-chain antibodies can also be bi-specific and/or humanized.
• a "Fab fragment” is comprised of one light chain and the variable and CH1 domains of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
• a "Fab' fragment” that contains one light chain and one heavy chain and contains more of the constant region, between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between two heavy chains is called a F(ab')2 molecule.
• a "F(ab')2" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between two heavy chains.
• buffer is used according to the art.
• An “equilibration buffer” is a buffer used to prepare the chromatography resin to receive the sample to be purified.
• a “loading buffer” refers to the buffer used to load the sample on the chromatography column or on a filter.
• a “wash buffer” is a buffer used to wash the resin. Depending on the mode of the chromatography, the “wash buffer” will allow the removal of the impurities (in bind/elute mode) or the collection of the purified sample (in flowthrough mode).
• An “elution buffer” refers to the buffer that is used to unbind the sample from the chromatographic material. This is possible thanks to the change of ionic strength between the load/wash buffers and the elution buffer.
• the purified sample containing the antibody will thus be collected as an eluate.
• the term "resin” or “chromatographic material” refer to any solid phase allowing the separation of the molecule to be purified from the impurities.
• Said resin or chromatographic material may be an affinity, an anionic, a cationic, an hydrophobic, or a mixed mode resin / chromatographic material. Units, prefixes and symbols are used according to the standards, i.e. International System of Units (SI).
• Anti-IL-6 receptor antibodies to be purified according to the process of the present invention are usually secreted by recombinant host cells into the culture medium, from which they can be recovered.
• the sample of antibodies to be purified according to the process of the present invention is preferably obtained (e.g. from the culture medium) at the time of harvest or post-harvest, should the sample be held for a certain amount of time before purification.
• the methods of the invention comprise a step of obtaining a sample of anti-IL-6 receptor antibodies to be purified.
• the sample can be for instance a crude harvest sample, a clarified harvest sample, or a post-harvest sample optionally equilibrated in an aqueous buffered solution.
• the present invention provides a method of purifying an anti-IL-6 receptor (anti-IL-6R) antibody from a sample containing the antibody and impurities, wherein the method comprises the following steps: (a) contacting the sample containing the antibody and the impurities with an affinity chromatography material under conditions such that the antibody binds to the resin and at least a portion of the impurities does not bind to the resin; (b) eluting the antibody from the affinity chromatography material, in order to obtain an eluate; (c) loading the eluate of step (b) onto a first mixed mode chromatography material under conditions such that the antibody does not bind to the resin and at least a portion of the remaining impurities binds to the resin; (d) recovering the flowthrough containing the antibody under conditions such that said recovered flowthrough contains a lower level of impurities than the eluate of step (b), (e) loading the recovered flowthrough containing the antibody of step (d) onto a second mixed mode chromatography material under
• the impurities are preferably selected from the group comprising or consisting of aggregates of the anti-IL-6 receptor antibody or fragments of said antibody or mixtures thereof, one or more of host cell proteins, endotoxins, viruses, nucleic acid molecules, lipids, polysaccharides, and any combinations thereof.
• the present invention provides a method of obtaining an anti-IL-6 receptor antibody in a monomeric form, wherein the method comprises the following steps: (a) contacting the sample containing the antibody in monomeric form, aggregated form and/or fragmented form with an affinity chromatography material under conditions such that the antibody binds to the resin and at least a portion of the aggregated form and fragmented form does not bind to the resin; (b) eluting the antibody in monomeric form from the affinity chromatography material, in order to obtain an eluate; (c) loading the eluate of step (b) onto a first mixed mode chromatography material under conditions such that the antibody in monomeric form does not bind to the resin and at least a portion of the remaining aggregated form and fragmented form bind to the resin; (d) recovering the flowthrough containing the antibody in monomeric form under conditions such that said recovered flowthrough contains a lower level of aggregated form and fragmented form than the eluate of step (b), (e) loading the recovered flow
• the anti-IL-6 receptor antibody to be purified according to the present invention is preferably a monoclonal antibody, which can be a chimeric antibody, a humanized antibody or a fully human antibody.
• the anti-IL-6 receptor antibody is a humanized monoclonal antibody, preferably tocilizumab.
• the humanized monoclonal antibody e.g. tocilizumab
• the cell lines also referred to as "recombinant cells” or "host cells" used in the invention are genetically engineered to express the anti-IL-6 receptor antibody of interest.
• the methods according to the invention are performed at room temperature (between 15°C and 25°C), except for the loading of step (a) which is typically performed between 2 and 8°C as the sample containing the antibody to be purified (here an anti-IL-6 receptor antibody of interest) is usually stored in cold conditions (between 2 and 8°C) after harvest as per standard procedure (see for instance Horenstein et al., 2003; [4]).
• the impurities to be removed in the context of the invention as a whole are selected from at least one of the group consisting of aggregates of the (monoclonal) antibody or fragments of said (monoclonal) antibody or mixtures thereof, one or more of host cell proteins, endotoxins, viruses, nucleic acid molecules, lipids, polysaccharides, and any combinations thereof.
• the invention is for removing aggregates and/or fragments of said (monoclonal) antibody.
• the recovered sample of step f), comprising the purified antibody comprises aggregates at a level of at least 90% lower than the level of aggregates in the sample of step (a).
• said recovered sample comprises fragments at a level of at least 40% lower than the level of fragments in the sample of step (a), and HCPs at a level of at least 90% lower or at least 95% lower than the level of HCPs in the sample of step (a) and below the typical acceptable limit of 100 ppm (such as lower than 50 ppm or 30 ppm).
• Protein A chromatography refers to the affinity chromatography technique using protein A, in which the protein A is usually immobilized on a solid phase.
• Protein A is a surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. There now exist various kinds of protein A of natural origin or produced recombinantly, possibly comprising some mutations as well. This protein has the ability to specifically bind the Fc portion of immunoglobulin such as IgG antibodies.
• Protein A chromatography is one of the most common affinity chromatography techniques used for purifying antibodies.
• the antibodies from a solution to be purified reversibly bind to the protein A, via their Fc portion. To the contrary (most of) the impurities flow through the column and are eliminated via washing steps. The antibodies thus need to be eluted from the column, or the affinity resin, in order to be collected for the next purification steps.
• the protein A chromatography material in step a) in the context of the present invention as a whole is selected for instance from the group, but not limited to, consisting of MABSELECTTM, MABSELECTTM SuRe, MABSELECTTM SuRe LX, AMSPHERETM A3, TOYOPEARL® AF-rProtein A- 650F, TOYOPEARL® AF-HC, PROSEP®-vA, PROSEP®-vA Ultra, PROSEP® Ultra Plus or ESHMUNO-A®.
• the Protein A ligand is immobilized on a matrix selected from the group consisting of dextran based matrix, agarose based matrix, polystyrene based matrix, hydrophilic polyvinyl ethyl based matrix, rigid polymethacrylate based matrix, porous polymer based matrix, controlled pore glass based matrix and any combination thereof.
• the Protein A ligand is immobilized on a membrane.
• the purpose of this step is to capture the antibodies (e.g. monoclonal antibodies such as toclizumab) present in the sample (e.g. a clarified harvest sample), concentrate them, and remove most of process-related impurities (such as HCPs, DNA, components of the cell culture broth).
• the antibodies e.g. monoclonal antibodies such as toclizumab
• the sample e.g. a clarified harvest sample
• process-related impurities such as HCPs, DNA, components of the cell culture broth.
• the sample, containing the anti-IL-6 receptor antibody of interest (e.g. tocilizumab), to be contacted with the affinity chromatography material in step a) is in an aqueous solution. It can be a crude harvest, a clarified harvest, or even a sample pre-equilibrated in an aqueous buffered solution. Before purification of the sample, the Protein A material has to be equilibrated. This equilibration is performed with an aqueous buffered solution. Suitable aqueous buffered solution (or buffers) include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers.
• the aqueous buffered solution for this step is preferably based on sodium acetate or sodium phosphate.
• the buffered solution is at a concentration in the range of or of about 10 mM to or to about 40 mM and a pH in the range of or of about 6.5 to or to about 8.0.
• the buffered solution is at a concentration in the range of or of about 15 mM to or to about 30 mM (such as between 20 and 30 mM) and its pH in the range of or of about 6.8 to or to about 7.5 (such as between 6.5 and 7.5).
• the concentration of the buffered solution is at or at about 15.0, 16.0, 17.0, 17.0, 18.0, 19.0, 20.0, 21 .0, 22.0, 23.0, 24.0 or 25.0 mM and its pH is at or at about 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4 and 7.5.
• the aqueous buffered solution to be used in one of the methods according to the invention can further comprise a salt at a concentration in the range of or of about 100mM to or to about 200mM, preferably at a concentration in the range of or of about 125 to 180 mM, such as of or of about 130, 135, 140, 145, 150, 155, 160, 165, or 170 mM.
• the salt is sodium chloride.
• the aqueous buffered solution comprises sodium phosphate at a concentration between 20 and 30 mM, a salt (e.g. sodium chloride) at a concentration between 100 and 200 mM, and/or has a pH in the range of 6.5 to about 7.5.
• the aqueous buffered solution can comprise sodium phosphate at a concentration of or of about 25 mM, sodium chloride at a concentration of or of about 150 mM, and has a pH of or of about pH 7.0 ⁇ 0.2.
• the skilled person will choose the appropriate conditions for equilibration and loading in order that the anti-IL-6 receptor antibody to be purified, such as tocilizumab, does bind to the affinity chromatography (e.g. Protein A chromatography) material. To the contrary, at least a part of the impurities will flow through the chromatography material (i.e. will not bind to the resin). For instance, the sample (e.g.
• clarified harvest sample) containing antibodies to be purified may be loaded at an amount of or of about 35-40g antibody per litre of packed affinity chromatography material, such as at an amount of or of about 37-40g antibody per litre of packed affinity chromatography material, such as at an amount of or of about 37g or 40g antibody per litre of packed affinity chromatography material.
• the affinity chromatography material is washed once or twice, with more of the same solution as the equilibration buffer or a different one, or a combination of both.
• suitable aqueous buffered solution include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers.
• the wash step is necessary to remove the unbound impurities.
• the wash is performed in one step, i.e. with one buffer.
• the wash buffer is an acetate buffer (such as a sodium acetate buffer) at a concentration in the range of or of about 40 mM to or to about 70 mM and a pH in the range of or of about 5.0 to or to about 6.0.
• the buffered solution is at a concentration in the range of or of about 45 mM to or to about 65 mM and its pH in the range of or of about 5.2 to or to about 5.8.
• the concentration of the buffered solution is at or at about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 mM and its pH is at or at about 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 and 5.8.
• the wash is performed in two steps with two different buffers.
• the first wash buffer is an acetate buffer (such as a sodium acetate buffer) at a concentration in the range of or of about 40 mM to or to about 70 mM and a pH in the range of or of about 5.0 to or to about 6.0.
• the buffered solution is at a concentration in the range of or of about 45 mM to or to about 65 mM and its pH in the range of or of about 5.2 to or to about 5.8.
• the concentration of the buffered solution is at or at about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 mM and its pH is at or at about 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 and 5.8.
• the second wash buffer is similar to the equilibration/loading buffer.
• the second wash buffer is a phosphate (e.g. sodium phosphate) buffer at a concentration in the range of or of about 15 mM to or to about 30 mM (such as between 20 and 30 mM) and having a pH in the range of or of about 6.8 to or to about 7.5 (such as between 6.5 and 7.5).
• the aqueous buffered solution to be used in one of the methods according to the invention can further comprise a salt.
• a salt Preferably, should a salt be present and should the method comprise a two- wash-step, said salt will be in a higher concentration in the first wash buffer than in the second wash buffer.
• the concentration of salt in the wash buffer (when 1-step only) or in the first wash buffer (when 2-steps), if any, is at a concentration in the range of or of about 1.0 M to or to about 2.0 M, preferably at a concentration in the range of or of about 1.25 to 1 .80 M, such as of or of about 1.3, 1 .35, 1.4, 1.45, 1.5, 1.55, 1.6, 1 .65, or 1.70 M.
• the concentration of salt in the second wash buffer is at a concentration in the range of or of about 100 mM to or to about 200 mM, preferably at a concentration in the range of or of about 125 to 180 mM, such as of or of about 130, 135, 140, 145, 150, 155, 160, 165, or 170 mM.
• Suitable salts include, but are not limited to, sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts.
• the salt is sodium chloride.
• the equilibration buffer comprises sodium phosphate at a concentration of or of about 25 mM
• a salt e.g. NaCI
• a first wash can be performed with a wash buffer comprising acetate (e.g.
• the anti-IL-6 receptor antibody such as tocilizumab
• elution buffer a solution that interferes with the binding of the affinity chromatography material to the constant domain of the antibody to be purified (here the anti-IL-6 receptor antibody).
• This elution buffer may include acetic acid, acetate glycine, citrate or citric acid.
• the buffered solution is an acetic acid buffer at a concentration in the range of or of about 40 mM to or to about 70 mM. More preferably, the buffered solution is at a concentration in the range of or of about 45 mM to or to about 65 mM.
• the concentration of the buffered solution is at or at about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 mM; such as acetic acid buffer at 55 mM.
• Elution may be performed by lowering the pH of the chromatography material and the antibody attached thereto.
• the pH of the elution buffer can be at or at about 4.5 or less, or at or at about 4.0 or less. It is preferably at or at about 2.8 to or to about 3.7, such as 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5 or 3.6; such as at pH 3.2 ⁇ 0.2.
• the elution buffer optionally includes a chaotropic agent.
• the elution i.e. elution of step (b)
• an elution buffer comprising acetic acid at a concentration of or of about 55 mM and a pH of 3.2 ⁇ 0.2.
• the mixed mode support according to the present invention refers to a chromatographic solid phase that involves a combination of two or more of the following functionalities (but not limited to): cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, or metal affinity. It thus comprises two different types of ligands.
• the solid phase can be a porous particle, nonporous particle, membrane, or monolith.
• the mixed mode chromatography material for step (c) involves a combination of two or more of cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, and hydrogen bonding; preferably involving at least anion exchange.
• the preferred mixed mode support for step (c) is selected from the group consisting of Capto-MMC, Capto-Adhere, Capto adhere Impress, MEP Hypercel and ESHMUNO HCX. It is preferably Capto-Adhere, having anion exchange properties.
• the eluate recovered after affinity chromatography (i.e. eluate of step (b)) is adjusted to a pH of 7.5 to 8.5, such as 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4 or 8.5.
• the conductivity of the eluate may also be adjusted, such as to or to about 10-20 mS/cm, such as to 15 ⁇ 0.5 mS/cm. Adjustment can be done with a concentrated solution of TRIS and/or NaOH for instance; such as using 2M Tris buffer.
• the aim is to have the eluate of step (b) at a pH and conductivity similar to the one under which step (c) is to be performed.
• step (c) for instance is to be performed at a pH of 8.0 ⁇ 0.2
• the eluate of step (b) has to be adjusted to a pH of 8.0 ⁇ 0.2.
• step (c) is to be performed with a salt (e.g. NaCI)
• same salt conditions will be used for the adjustment.
• the eluate e.g. after adjustment
• the first mixed mode chromatography material is equilibrated with an aqueous buffered solution (equilibration buffer).
• aqueous buffered solution include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers.
• the buffered solution is a phosphate (e.g. sodium phosphate) buffer.
• the buffered solution (e.g.
• a sodium phosphate buffer is preferably at a concentration in the range of or of about 20 mM to or to about 60 mM and a pH in the range of or of about 7.5 to or to about 8.5.
• the buffered solution is at a concentration in the range of or of about 30 mM to or to about 50 mM and its pH in the range of or of about 7.5 to or to about 8.5.
• the concentration of the buffered solution is at or at about 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41 .0, 42.0, 43.0, 44.0 or 45.0 mM and its pH is at or at about 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2 or 8.3.
• the aqueous buffered solution to be used in one of the methods according to the invention can further comprise a salt at a concentration in the range of or of about 75 mM to or to about 125 mM, preferably at a concentration in the range of or of about 85 to 120 mM or 85 to 1 10 mM, such as of or of about 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 mM.
• Suitable salts include, but are not limited to, sodium chloride and/or potassium chloride; preferably sodium chloride.
• the first mixed mode chromatography material is equilibrated with an equilibration buffer that is an aqueous buffered solution comprising sodium phosphate at a concentration between 30 and 50 mM, a salt (e.g. NaCI) at a concentration between 80 and 120 mM, and a pH in the range of 7.5 to 8.5.
• the equilibration buffer will also be used to "push" the unbound antibodies/proteins (anti-IL-6 receptor antibody) in the flowthrough, in order to recover said purified antibodies/proteins (step d).
• the skilled person will chose the appropriate conditions for this first mixed mode chromatography step in order that the anti-IL-6 receptor antibody to be purified, such as tocilizumab, does not bind to the first mixed mode chromatography material, i.e. in order that it flows through the chromatography material. Said flowthrough is recovered at the bottom of the column. To the contrary, at least a part of the impurities bind to the chromatography material.
• the equilibration buffer for the first mixed mode chromatography step comprises sodium phosphate at a concentration of or of about 40 mM, a sodium chloride at a concentration of or of about 95 mM and a pH of 8.0 ⁇ 0.2.
• the first mixed mode chromatography material can be loaded with eluate at an amount of or of about 80 to or to about 120g anti-IL-6 receptor antibody(e.g. toclizumab) per litre of packed resin; such as at an amount of or of about 1 10 g antibody per litre of packed resin.
• the preferred mixed mode support for the second mixed mode chromatography step (step (e)) is selected from the group consisting of hydroxyapatite- based ligand (or resin), hydroxyfluorapatite-based ligand (or resin) or fluorapatite-based ligand (or resin).
• An hydroxyapatite-based resin is a mixed mode support comprising a mineral of calcium phosphate with the structural formula (Ca 5 (P0 4 ) 3 0H) 2 . Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity. Hydroxyapatite-based resins are commercially available in various forms, including but not limited to ceramic composite forms. Commercial examples of ceramic hydroxyapatite include, but are not limited to CHTTM Type I and CHTTM Type II. Ceramic hydroxyapatites are porous particles and can have various diameters, for instance about 20, 40, and 80 microns.
• a fluorapatite-based resin is a mixed mode support comprising an insoluble fluoridated mineral of calcium phosphate with the structural formula Ca 5 (P0 4 )3F or Ca-io(P0 4 )6F 2 . Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity.
• Fluorapatite-based resins are commercially available in various forms, including but not limited to ceramic composite forms. Commercial examples of ceramic fluorapatite include, but are not limited to CFTTM Type I and CFTTM Type II. Ceramic fluorapatites are spherical porous particles and can have various diameters, for instance about 10, 20, 40, and 80 microns.
• a hyd roxyfl uo ra pa ti te- ba sed resin a mixed mode support comprising an insoluble hydroxylated and an insoluble fluoridated mineral of calcium phosphate with the structural formula Ca10(P04)6(OH)x(F)y. Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity. Hydroxyfluorapatite is commercially available in various forms, including but not limited to ceramic, crystalline and composite forms. Composite forms contain hydroxyfluorapatite microcrystals entrapped within the pores of agarose or other beads.
• Ceramic Hydroxyfluorapatite resin is the MPC Ceramic Hydroxyfluorapatite ResinTM, with a structural formula (Caio(P04)6(OH) 1 5 (F)o.5), It is based on the ceramic apatite Type I (40 ⁇ ⁇ ) mixed-mode resin.
• the flowthrough recovered after the first mixed mode chromatography is adjusted to a pH of 7.0 to 8.5 such as 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. Adjustment can be done with a concentrated solution of TRIS and/or NaOH for instance.
• the aim is to have the flowthrough of step (d) into conditions suitable for the load on the second mixed mode chromatography. If step (e) for instance is to be performed at a pH of 7.5 ⁇ 0.2, the flowthrough of step (d) has to be adjusted to a pH of 7.5 ⁇ 0.2. This step of adjustment can be performed together with a concentration step, such as via tangential flow filtration (TFF).
• TRIS tangential flow filtration
• a filtration step can be added before the second mixed mode chromatography.
• Other adjustments that are needed relate to salts (e.g. NaCI) and NaP0 4.
• the second mixed mode chromatography material is equilibrated with an aqueous buffered solution (equilibration buffer).
• the flowthrough recovered after the first mixed mode chromatography step (step (d)) is equilibrated prior to loading onto the second mixed mode chromatography material (of step (e)) with an aqueous buffered solution.
• Suitable aqueous buffered solution include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers.
• the buffered solution is a phosphate (e.g. sodium phosphate) buffer.
• the buffered solution e.g. a sodium phosphate buffer
• the buffered solution is preferably at a concentration in the range of or of about 1 mM to or to about 20 mM and a pH in the range of or of about 7.0 to or to about 8.0. More preferably, the buffered solution is at a concentration in the range of or of about 2 mM to or to about 15 mM and its pH in the range of or of about 7.2 to or to about 7.8.
• the concentration of the buffered solution is at or at about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0 mM and its pH is at or at about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 and 7.8.
• the aqueous buffered solution to be used in one of the methods according to the invention can further comprises a salt at a concentration in the range of or of about 100mM to or to about 250mM, preferably at a concentration in the range of or of about 130 to 200 mM, such as of or of about 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190 or 195 mM.
• Suitable salts include, but are not limited to, sodium chloride and/or potassium chloride; preferably sodium chloride.
• the second mixed mode chromatography material is equilibrated with an equilibration buffer that is an aqueous buffered solution comprising sodium phosphate at a concentration between 1 and 10 mM, optionally a salt (e.g. NaCI) at a concentration between 130 and 200 mM, and a pH in the range of 7.0 to 8.0.
• an equilibration buffer that is an aqueous buffered solution comprising sodium phosphate at a concentration between 1 and 10 mM, optionally a salt (e.g. NaCI) at a concentration between 130 and 200 mM, and a pH in the range of 7.0 to 8.0.
• the equilibration buffer will also be used to "push" the unbound antibodies/proteins in the flowthrough, in order to recover said purified antibodies/proteins (step f).
• the skilled person will chose the appropriate conditions for this second mixed mode chromatography step in order that the anti-IL-6 receptor antibody to be purified, such as tocilizumab, does not bind to the first mixed mode chromatography material, i.e. in order that it flows through the chromatography material. Said flowthrough is recovered at the bottom of the column. To the contrary, at least a part of the impurities bind to the chromatography material.
• the second mixed mode chromatography step can be performed in an aqueous buffered solution comprising 5 mM sodium phosphate, 170 mM sodium chloride and a pH of 7.5 ⁇ 0.2. Loading is performed in the same condition.
• the second mixed mode chromatography material can be loaded with eluate at an amount of at least 60g anti-IL-6 receptor antibody (e.g. toclizumab) per litre of packed resin; such as at an amount of or of about 60 g antibody per litre of packed resin.
• the second mixed mode chromatography material can be loaded with eluate at an amount of or of about 80 to or to about 120g anti-IL-6 receptor antibody (e.g. toclizumab) per litre of packed resin; such as at an amount of or of about 1 10 g antibody per litre of packed resin.
• the method according to the present invention comprises a step of virus inactivation.
• This step is preferably performed between the affinity chromatography step (after elution of the antibody) and the first mixed mode chromatography step. It is called step (b').
• the eluate recovered after affinity chromatography step i.e. the eluate of step (b)
• the pH to be reached during adjustment is preferably in a range of or of about 3.0 to or to about 4.5, even preferably in a range of or of about 3.2 to or to about 4.0, such as 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0; typically pH 3.5 ⁇ 0.2.
• the concentration of the salt in the acidic aqueous solution used for adjustment is at or at about 1.5 to or to about 2.5.
• the concentration of the salt in the acidic aqueous solution is at or at about 1.7 to or to about 2.3, such as 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, or 2.3 M.
• the preferred acidic aqueous solution is acetic acid; such as at a concentration of or of about 2M.
• the eluate is adjusted to pH 3.5 ⁇ 0.2 using 2M acetic acid. The resulting adjusted eluate is typically incubated for about 60 ⁇ 15 min.
• the material is then neutralized with a concentrated neutral aqueous solution.
• the pH to be reached during neutralization is preferably in a range of or of about 4.5 to or to about 6.5, should the neutralized sample be held before step (c), even preferably in a range of or of about 4.8 to 5.6 such as 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5 or 5.6.
• pH to be reached during neutralization will be the same pH as the one that will be used for step (c), i.e. from 6.5 to 8.5 (such as from 7.5 to 8.5, such as pH 8 ⁇ 0.2).
• the concentration of the salt in the aqueous solution used for neutralization is at or at about 1 .0 to or to about 2.5.
• the concentration of the salt in the neutral aqueous solution is at or at about 1.0 to or to about 2.0, such as 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 M.
• the preferred neutral aqueous solution is Tris base; such as at 2M.
• the virus-inactivated material is neutralized to pH 5.2 ⁇ 0.2 using 2M Tris base.
• Various filtration steps can be added in the purification process. Such steps may be needed to further eliminate impurities but can also be used to concentrate the sample to be purified before the next chromatographic step or to change the buffer before the next chromatographic step.
• a filtration step can be performed just before the first mixed mode chromatography.
• This filtration step is preferably performed with a depth filter. Said step can be performed in line with the first mixed mode chromatography.
• a filtration step such as a depth filtration, can be included during the process.
• This step can for instance be added just before the first mixed mode chromatography, as described in Example 2.
• Tangential Flow Filtration (TFF) can also be performed during the purification procedure.
• TFF Tangential Flow Filtration
• a TFF can be performed just before step (e).
• Such filtration step can be performed with the equilibration buffer that will be used for the second mixed mode chromatography. This will allow the flowthrough not only to be concentrated but also to be in such a condition to be ready for the next chromatographic step.
• the TFF step can be performed in an aqueous buffered solution comprising 5 mM sodium phosphate, 170 mM sodium chloride, pH 7.5 ⁇ 0.2.
• SEQ ID NO 1 light chain of tocilizumab, amino acid sequence
• SEQ ID NO 2 heavy chain of tocilizumab, amino acid sequence
• the anti-IL-6R antibody used in the example is tocilizumab (see SEQ ID Nos.1-2). It was produced in CHO-K1 cells. Cells were cultured in fed-batch culture. They were incubated at 36.5°C, 5% de CO2, 90% humidity and shaken at 320rpm. Each of the fed-batch culture lasted 14 days. II. Analytical methods
• HCPs level in ppm is calculated using the HCPs level determined in ng/mL divided by the imAb concentration determined by UV absorbance (mg/mL).
• HMW Content in aggregates
• LMW Low Molecular weight
• Example 1 Tocilizumab purified according to a standard process
• the full purification process was performed at room temperature (15-25°C) except for the load step of the Protein A step, as the clarified harvest was stored at 2-8°C before purification.
• Tocilizumab was purified according to standard purification steps including "protein A chromatography” followed by a first "ion exchange chromatography” in bind elute followed by a second “ion exchange chromatography” in flowthrough (also called polishing step).
• Example 2 - Tocilizumab purified according to the process of the invention The full purification process was performed at room temperature (15-25°C) except for the load step of the Protein A step, as the clarified harvest was stored at cold temperature before purification (i.e. at 2-8°C).
• the new process, according to the invention had been used to improve the purification scheme for tocilizumab.
• the main steps for this new process were:
• Protein a step was performed on a Prosep Ultra Plus® resin (from Merck Millipore), with a target bed height of 20 ⁇ 2cm. This step was performed under the following conditions:
• Wash I >5 BV of a solution comprising 55mM sodium Acetate, 1 .5M NaCI, pH 5.5.
• Wash II >3 BV of a solution comprising 25mM NaPI+150mM NaCI, pH 7.0.
• the Protein A eluate was adjusted to pH 3.5 ⁇ 0.2 by addition of 2M acetic acid solution under stirring. Once the target pH was reached, the agitation was stopped and the acidified eluate was incubated for 60 ⁇ 15 min. At the end of the incubation, the material was neutralized to pH 5.2 ⁇ 0.2 by addition of 2M Tris Base solution under stirring. The resulting eluate (neutralized eluate) can be stored at least 3 months at 2-8°C.
• the neutralized eluate was adjusted to pH 8.0 ⁇ 0.2 with 2M Tris and its conductivity was increased to 15.0 ⁇ 0.5 mS/cm with 3M NaCI. This adjusted eluate was then submitted to depth filtration in-line with mixed mode chromatography on Capto Adhere® (from GE Healthcare) as follows:
• the depth filter (Millistack Pod from Merck Millipore) was connected to the purification system in front of the chromatography column.
• wash 4BV of 40mM NaPI, 93mM NaCI, pH 8.0. Stop collection of the flowthrough containing the purified tocilizumab.
• the flowthrough from mixed mode chromatography 1 was concentrated via TFF, on a Pellicon 3 Ultracel® 30kDa membrane (from Merck Millipore). This step allowed also to exchange the buffer into conditions suitable for the load of the Fluorapatite chromatography step, on a CFT Ceramic Fluorapatite® Type II (40um) (from Bio-Rad).
• the TFF step was performed as follows:
• the mixed mode chromatography 2 step was performed as follows:

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• 2018
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